Ascorbate is a required component in the diet of humans. Ascorbate is the agent which prevents scurvy, and is known to take part in several biological reactions including the formation of collagen, the formation of neurotransmitters, and the degradation of tyrosine (Jaffe G. (1984) in: Handbook of Vitamins (Machlin L. (ed.), Marcel Dekker, Inc. pp. 199-244; Flier J. S. and Underhill L. H. (1986) N. Engl. J. Med. 314:892-902).
Ascorbate is easily oxidized through a free radical intermediate (semi-dehydroascorbate) to form dehydroascorbate, providing electrons to be used in various reactions. Transition metals, particularly Fe(III) and Cu(II) are well described catalysts for oxidizing ascorbate, producing hydrogen peroxide and hydroxyl radicals from molecular oxygen in the process (Miller D. M. et al. (1990) Free Radical Biol. & Med. 8:95-108; Taquil M. M. and Martell A. E. (1967) J. Am. Chem. Soc. 89:4176-4185).
Ascorbate is well-known as an antioxidant in vitro, being used, for example, to prevent the oxidation of reduced folates into oxidized forms in red cell folate assays (Rothenberg S. P. et al. (1972) New Engl J. Med. 286:1335-1339) or as a reducing agent in myeloperoxidase reactions (Marquez L. A. et al. (1990) J. Biol. Chem. 265:5666-70). Furthermore, ascorbate plays an important role as a reducing agent in vivo, as shown by the augmentation caused by ascorbate on the formation of reduced folates from oxidized folates by liver (Nichol C. A. and Welch A. D. (1950) Proc. Soc. Exp. Med. 74:52-55). Recent studies suggest that ascorbate may be the primary extracellular antioxidant in plasma (Frei B. et al. (1989) PNAS (USA) 86:6377-6381) and that other physiologic reducing compounds such as vitamin E, are maintained in the reduced state at the expense of ascorbate (Maguire J. J. et al. (1989) J. Biol. Chem 264:21462-5). Ascorbate has been found to be more effective than glutathione at detoxifying the acetaminophen phenoxyl free radical (Ramakrishna-Rao D. N. et al. (1990) J. Biol. Chem. 265:844-7). Endogenously generated oxidants are thought to be important in carcinogenesis (Weitzman S. A. and Gordon L. I. (1990) Blood 76:655-663), giving ascorbate a potential physiologic role in cancer prevention (Stahelin H. B. et al. (1987) in: Third Conference on Vitamin C (Burns J. et al. (eds) 498, pp. 124-131).
Assays for ascorbate are important to better determine biochemical and physical roles for ascorbate in health and disease. However, the results of currently existing assays must be interpreted with caution since ascorbate is unstable in solution, with measurable degradation in aqueous systems occurring within minutes to hours (Washko P. W. et al (1989) Anal. Biochem. 181:276-82), presumably due to molecular oxygen and traces of contaminating catalytic metals (Buettner G. R. (1988) J. Biochem. Biophys. Methods 16:27-40). Aqueous ascorbate instability is illustrated in the Examples and Table 1 herein. The inventors hereof have found that such degradation also occurs in body fluids. Because body fluids can be stored for days or perhaps weeks prior to ascorbate assay, the measured ascorbate can bear little relation to the in vivo ascorbate concentration As also illustrated in the Examples, freezing may slow the ascorbate degradation process in serum or plasma, but does not arrest it. The extent of ascorbate degradation in collected body fluid is related to the nature of the fluid and the methods of collection and storage. Degradation can vary significantly from one body fluid sample to another.
Inaccurate in vivo ascorbate assays have practical significance, for example, in epidemiologic studies which attempt to correlate plasma ascorbate levels with common fatal human diseases such as cancer (Stahelin H. B. et al (1987) in: Third Conference on Vitamin C (Burns J. et al. (eds) 498, New York Academy of Sciences, pp. 124-131) and ischemic heart disease (Gey K. F. et al. (1987), Id. at pp. 110-120). If changes in ascorbate are occurring in vitro with plasma storage, the epidemiologic data would be subject to error.
Recently, ascorbate assay methods involving HPLC (which provides specificity) and ultraviolet absorption or changes in electrical current or potential (for quantitation) have been described. See Frei B. et al. (1989) PNAS (USA) 86:6377-6381; Washko P. et al. (1989) J. Biol. Chem. 264:13996-19002; Washko P. W. et al. (1989) Anal. Biochem. 181:276-82; and Honegger C. G. et al. (1986) J. Chromatography 381:249-258). However, none of these methods use an internal standard to quantitate the loss of ascorbate in vitro during sample processing and preparation. Other methods, including electron impact mass spectroscopy (Ng Y-C et al (1985) Biochem. Pharm. 34:2525-2530), laser desorption mass spectroscopy (McMahon J. M (1985) Anal. Biochem 147:535-545), and gas chromatography/mass spectroscopy (Knaack D. and Podleski T. (1985) PNAS (USA) 82:575-579) have been used to definitively identify ascorbate, but have not been use to quantitate ascorbate.
Therefore, a need exists for an ascorbate assay that accurately determines loss of ascorbate in vitro during sample storage or processing so that in vivo ascorbate can be calculated.
It would be particularly advantageous if said method for accurately determining in vivo ascorbate concentration could also be used to quantitate the redox potential of body fluids. Quantitation of the redox potential of blood or other body fluids can be a useful means of measuring the oxidative stress of an individual. Oxidative stress can develop, for example, in individuals undergoing oxygen treatment, such as premature infants or persons that have recently undergone surgery. The resulting oxygen toxicity (e.g., adult respiratory distress syndrome (ARDS)) is characterized by the depletion of ascorbate and other reducing agents in the blood or other body fluids. Since the diet is the only source of ascorbate for humans, adequate supplementation to assure normal ascorbate blood levels would be necessary to prevent these complications.
The redox potential of human blood is determined by several processes including the following redox reactions: ascorbate to dehydroascorbate and other metabolites; homocysteine/cysteine to oxidized disulfides; reduced glutathione to oxidized glutathione; Vitamin E to oxidized Vitamin E; and Vitamin A to oxidized Vitamin A. The most sensitive of these indicators of the redox potential may be ascorbate since ascorbate, relative to the other redox species, is the most readily oxidized on exposure to air. Ascorbate's antioxidant function has been found to protect lipids, .alpha.-tocopherol, urate and bilirubin from peroxidation by aqueous peroxyl radicals. See Frei B. et al. (1989) PNAS (USA) 86:6377-6381.
Although oxidant stress status is an important indicator of potential disease, (see, e.g., Smith (1991) Free Radicals in Biology and Medicine 10:217-224; and Pryor and Godber (1991), Free Radicals in Biology and Medicine 10:171-184), no method is presently known to the Applicants for measuring the redox potential of body fluids using ascorbate and its oxidation products. Washko P. et al. (1989) J. Biol. Chem. 264:18996-19002, describe the direct measurement of ascorbate and the indirect determination of dehydroascorbate in human neutrophils using high performance liquid chromatography and coulometric electrochemical detection. Dhariwal et al. (1990) Anal. Biochem 189:18-23 measure DHA indirectly by assaying for ascorbic acid, reducing DHA to ascorbic acid, then measuring total ascorbic acid. These methods are not, however, used for the measurement of redox potential. Additionally, Washko and Dhariwal do not use an internal standard, and thus do not account for loss of ascorbate or its metabolites during sample storage. Further, these workers do not measure dehydroascorbate directly and do not measure other ascorbate metabolites at all, and their method is therefore believed to not be an accurate method of measuring in vivo ascorbate concentration, in vivo concentrations of ascorbate metabolites, or redox potential.
Thus, a method for the accurate determination of the in vivo concentration of ascorbate and its metabolites would not only provide an accurate ascorbate assay for epidemological and other studies, but also a sensitive method of measuring an individual's body fluid redox potential. Such method would be useful in epidemiologic studies linking oxidative stress, oxidative injury and disease to in vivo ascorbate metabolism.